Forschungsprofil Dr. Özkan Yildiz

Dr. Özkan Yildiz
  • Ph.D. Max-Planck-Institute of Molecular Physiology, Dortmund, 2000-2003
  • Dr. rer. nat. (Chemistry), Ruhr-University Bochum, July 2003
  • Postdoc at the Max Planck Institute of Molecular Physiology, Dortmund, 2003-2004
  • Postdoc at the Max Planck Institute of Biophysics, 2004
  • Project Leader at the Institute, since 2008

Structure and functions of proteins involved in membrane transport

The group's main focus is the 3D crystallization, X-ray structure determination and functional characterization of proteins involved in transport and signalling across the membrane. In combination with electron microscopic methods like electron cryo-microscopy or single particle analysis, we try to get insights into the structure and mechanisms of the studied proteins. The monomeric porin OmpG from the E. coli outer membrane, the sodium proton antiporter NhaP1 and GlnK1, the regulator of the ammonium channel Amt1 from Methanocaldoccus jannaschii are among the investigated proteins.

Fig. 1: 3D X-ray structures of OmpG in the open and closed form. The X-ray structure of E. coli OmpG crystallized at pH 8.0 shows the channel in open conformation (A, B). At pH 5.6 folding of one loop (L6) blocks the channel (C, D).

OmpG: The outer membrane porin from E. coli is a unusual protein. Unlike classical trimeric porins, the functional unit of OmpG is monomeric. First structural informations were achieved from projection maps (Behlau et al., JMB 2001) obtained by electron cryo-microscopy of two-dimensional (2D) crystals. The projection maps showed a circular shaped monomeric β-barrel with a diameter of around 25 Å. Initial secondary structure predictions of OmpG suggested 16 β-strands, but the diameter of the β-barrel in the projection maps agrees better with 14 β-strands. To investigate OmpG in detail, we expressed OmpG as inclusion bodies in E. coli, purified it by unfolding and refolding, and crystallized the refolded protein in 3D.
Two different crystal forms, grown at different condition (pH 5.6 and pH 8.0) could be optimized for data collection. Using the initial structure determined by SeMet-MAD technique, the structures of both crystal forms were solved by the molecular replacement method. OmpG is indeed a 14-stranded β-barrel and a pore-forming monomer. A long extracellular loop assumes two distinct, well-defined conformations, apparently in response to the pH of the medium. At neutral pH it projects into the extracellular medium, leaving the pore wide open, whereas at low pH it folds across the pore channel and blocks it, suggesting a direct role in pH-dependent pore gating.  (more details..)

Figure 2: Top view (A) and side view (B) of the 3D map of NhaP1 from M. jannaschii obtained by electron crystallography of 2D crystals. The six-helix bundle and the group of transmembrane helices near the dimer interface in (A) are shown on a green or yellow background, respectively.

NhaP1: Na+/H+ antiporters (CPAs) are crucial for maintaining the balance of Na+ and H+ in the cell. Both ions are vital in physiology and bioenergetics. Na+/H+ antiporters are found in cytoplasmic and organellar membranes of all living cells, including animals, plants, and prokaryotes.

The first insights into the molecular architecture of Na+/H+ antiporters came from electron crystallography of two-dimensional (2D) crystals of E. coli NhaA (Williams et al., 1999 and Williams, 2000). The 3.5 Å X-ray structure of NhaA at pH 4.0 revealed a unique fold where the trans-membrane helices (TMHs) IV and XI form pairs of half helices connected by short stretches of unwound polypeptide and gave new insights in to the mechanism of ion exchange and pH regulation (Hunte et al., 2005). Recent studies of 2D crystals of NhaA at different conditions revealed subtle, but well-defined and distinguishable conformational changes induced by pH changes or substrate ions (Appel et al, 2009).

Apart from NhaA, NhaP1 of Methanococcus jannaschii is currently the only other member of the CPA superfamily for which structural information is available. Sequence comparisons show that MjNhaP1, shares 18-21% identity to eukaryotic transporters like SOS1 (Arabidopsis thaliana) and NHE1 (Homo sapiens), whereas the sequence similarity to E. coli NhaA is only 10-16%. Hydrophobicity analysis have suggested 13 rather than 12 TMHs in MjNhaP1 (Hellmer et al., 2003). The activation of this antiporter occurs between a pH value 6 and 7, a property shared by the human homologue, NHE1. By contrast, the E. coli NhaA becomes active at a pH above 7 and is switched off below it.

Recently, we solved the structure of NhaP1 at 7Å resolution and we could show that the additional helix plays a role in protein activity (more details…).

Fig. 3: High resolution X-ray structures of GlnK. Structure of GlnK1 with bound Mg-ATP at 1.2 Å resolution (A, C). All three monomers (C) of the trimeric GlnK1 (A) bind ATP and Mg. Mg-ATP (red) fixes the T-loop in the compact conformation through main-chain interactions with the ATP -phosphate and hydrogen bonds to Mg-coordinated water molecules. Structure of GlnK1 in the absence of added Mg-ATP at 2.1 Å resolution (B). Without added Mg-ATP, occasional sites at the interface between two adjacent monomers (blue and green) are occupied with ADP (red) from the expressing cells. The T-loop is in an extended conformation, with arginines 45, 47 and 49 and Tyr51 at the tip (B).
Fig. 4: Surface potential presentation of the A. fulgidus Amt1 trimer and M. jannaschii GlnK1. Facing views of interacting surfaces of Amt1 with red, negatively charged channel vestibules (A) and GlnK1 in the absence of Mg-ATP with blue, positively charged contact surface and T-loops (B). Side views of Amt1 in the membrane and the GlnK1 trimer with extended T-loops (C). Model of the binary Amt1/GlnK1 complex with the T-loops docked into the cytoplasmic pore vestibules (D). The Mg-ATP binding pocket on GlnK1 is visible as a dark blue, positively charged cavity on the side of the complex. With bound Mg-ATP, the charge on the T-loops is compensated and they are fixed in the compact conformation, so they cannot insert into the vestibules.

Amt1-GlnK1: Biosynthesis of macromolecules requires reduced nitrogen. While higher eukaryotes usually take up nitrogen in the form of amino acids, the prevalent source for microorganisms is either uncharged ammonia (NH3) or the ammonium ion (NH4+). Free diffusion of ammonia through the membrane is uncontrolled, and at low or neutral pH, it is too slow. Therefore, many microorganisms have evolved systems for highly efficient and strictly regulated ammonium uptake. Members of the ammonium uptake (Amt) family of membrane proteins are found in bacteria, archaea, fungi, and plants. Mammalian homologues are the Rhesus glycoproteins. The high-resolution crystal structures of AmtB from Escherichia coli (Khademi et al., 2004; Zheng et al., 2004) and Amt1 from the hyperthermophilic archaeon Archaeoglobus fulgidus (Andrade et al., 2005) show that the Amt proteins are trimers, with 11 membrane-spanning helices in the monomer forming a bundle around a hydrophobic channel. The X-ray structures imply that Amt proteins act as channels enabling controlled uptake of ammonia rather than active transport of ammonium ions (Khademi et al., 2004). Each monomer has an ammonium-binding site at the extracellular pore entrance that might deprotonate the ammonium ion for passage through the channel. On the cytoplasmic side ammonia would again accept a proton at physiological pH to form the ammonium ion.

Future  Projects

OmpG: Introduction of mutations on the basis of the structural informations should help to characterize the functional properties of OmpG and should help also to generate channels with altered functions.

NhaP1: High-resolution X-Ray structure of NhaP1 and homologues. Capture of NhaP1 in different conformational states. Further biochemical characterization.

Amt1-GlnK1: High-resolution X-ray structure of the AmtB-GlnK complex confirmed our model based on EM single particle analysis and X-ray structures of GlnK1.

 

Internal Collaborations:

Cristina Paulino

Dr. Thomas Meier

Dr. Denys Pogoryelov

 

 
External Collaborations:

Prof. Dr. Werner Mäntele and Filiz Korkmaz (Institute of Biophysics, Johann Wolfgang Goethe University, Frankfurt am Main)
Prof. Dr. Christian Herrmann and Mark Wehner (Physikalische Chemie I, Arbeitsgruppe Protein-Interaktionen, Ruhr-University,  Bochum)
Prof. Dr. Daniel J. Müller (Biotechnology Center, TU Dresden)
Prof. Dr. Trinad Chakraborty and Dr. Torsten Hain (Institute of Medical Microbiology, Justus Liebig University, Gießen)

 

Grant  Support

The OmpG-Project is supported by the Deutsche Forschungsgemeinschaft, DFG,
DFG-Project YI 96/3-1

 

 

Invited  Lectures

9th International Conference on Biology and Synchrotron Radiation (13-17 August 2007, Manchester, England)

 

Publications

Goswami P., Paulino C., Hizlan D., Vonck J., Yildiz Ö., Kühlbrandt W.
Structure of the archaeal Na+/H+ antiporter NhaP1 and functional role of transmembrane helix 1.
EMBO J
. 30 439-49 (2011).

Damaghi M., Sapra K.T., Köster S., Yildiz Ö., Kühlbrandt W., Muller D.J.
Dual energy landscape: the functional state of the β-barrel outer membrane protein G molds its unfolding energy landscape.
Proteomics
. 10 4151-62 (2010).

Pogoryelov D., Krah A., Langer J.D., Yildiz Ö., Faraldo-Gómez J.D., Meier T.
Microscopic rotary mechanism of ion translocation in the F(o) complex of ATP synthases.
Nat Chem Biol
. 6 891-9 (2010)

Preiss L., Yildiz Ö., Hicks D.B., Krulwich T.A., Meier T.
A new type of proton coordination in an F(1)F(o)-ATP synthase rotor ring.
PLoS Biol
. 8 (8) e1000443 (2010).

Korkmaz-Özkan F., Köster S., Kühlbrandt W., Mäntele W., Yildiz Ö.
Correlation between the OmpG secondary structure and its pH-dependent alterations monitored by FTIR.
J Mol Biol
. 401 (1) 56-67 (2010).

Damaghi M., Bippes C., Köster S., Yildiz Ö., Mari S.A., Kühlbrandt W., Muller D.J.
pH-dependent interactions guide the folding and gate the transmembrane pore of the beta-barrel membrane protein OmpG.
J Mol Biol
. 397 (4) 878-82 (2010).

Mari S.A, Köster S., Bippes C.A., Yildiz Ö., Kühlbrandt W., Muller D.J.
pH-induced conformational change of the beta-barrel-forming protein OmpG reconstituted into native E. coli lipids.
J Mol Biol
. 396 (3) 610-6 (2010).

Hennig, S., Strauss, H., Vanselow, K., Yildiz, Ö., Schulze, S., Arens, J., Kramer, A., and Wolf, E.
Structural and Functional Analyses of PAS Domain Interactions of the Clock Proteins Drosophila PERIOD and mouse PERIOD2.
PLoS Biol. 7 (4) e1000094 (2009).

Köster, S., Kühlbrandt, W., and Yildiz, Ö.
Purification, crystallization and preliminary X-ray diffraction analysis of the FeoB G domain from Methanococcus jannaschii.
Acta Cryst. F65, 684-687 (2009).

Köster, S., Wehner, M., Herrmann, C., Kühlbrandt, W., and Yildiz, Ö.
Structure and function of the FeoB G-domain from Methanococcus jannaschii.
J Mol Biol. 392 (2) 405-419 (2009).

Pogoryelov, D., Yildiz, Ö., Faraldo-Gómez, J. D., and Meier, T.
High-resolution structure of the rotor ring of a proton-dependent ATP synthase.
Nat Struct Mol Biol. 16 (10) 1068-1073 (2009).

Sapra, K. T., Damaghi, M., Köster, S., Yildiz, Ö., Kühlbrandt, W., and Muller, D. J.
One β Hairpin after the Other: Exploring Mechanical Unfolding Pathways of the Transmembrane β-Barrel Protein OmpG.
Angew Chem Int Ed. 48 (44) 8306-8308 (2009).

Korkmaz, F., Köster, S., Yildiz, Ö., and Mäntele, W.
The Role of Lipids for the Functional Integrity of Porin: An FTIR Study Using Lipid and Protein Reporter Groups.
Biochemistry 47 12126-12134 (2008).

Stieglitz, B., Bee, C., Schwarz, D., Yildiz, Ö., Moshnikova, A., Khokhlatchev, A., and Herrmann, C.
Novel type of Ras effector interaction established between tumour suppressor NORE1A and Ras switch II.
EMBO J. 27 1995-2005 (2008).

Yildiz, Ö., Kalthoff, C., Raunser, S., and Kühlbrandt, W.
Structure of GlnK1 with bound effectors indicates regulatory mechanism for ammonia uptake.
EMBO J. 26 589-599 (2007).

Yildiz, Ö., Vinothkumar, K. R., Goswami, P., and Kühlbrandt, W.
Structure of the monomeric outer-membrane porin OmpG in the open and closed conformation.
EMBO J. 25 3702-3713 (2006).

Yildiz, Ö., Doi, M., Yujnovsky, I., Cardone, L., Berndt, A., Hennig, S., Schulze, S., Urbanke, C., Sassone-Corsi, P., and Wolf, E.
Crystal structure and interactions of the PAS repeat region of the Drosophila clock protein PERIOD.
Mol. Cell. 7 69-82 (2005).

 

Ph. D. Thesis:
Panchali Goswami, "The three dimensional structure of the Na+/H+ antiporter from Methanocaldococcus jannaschii by electron crystallography".
Johann Wolfgang Goethe-Universität Frankfurt am Main, 2009.

 

Diploma Thesis:
Tim Jacobs, Biochemische und strukturelle Untersuchungen von Proteinen des feo-Operons. Johannes Gutenberg-Universität Mainz, 2009.

Bahman Ossuli, Fachhochschule Gießen-Friedberg, 2010

Kai Holländer, Strukturelle Charakterisierung ausgewählter archaealer Na+/H+-Antiporter. Fachhochschule Gießen-Friedberg, 2010

Kontaktinformationen:

Max-Planck-Institut für Biophysik

Dr. Özkan Yildiz
Department of Structural Biology

Tel.: +49 (0)69 6303-3051
Fax:  
+49 (0)69 6303-3002

E-Mail: oezkan.yildiz(at)biophys.mpg.de

Yildiz - Gruppenmitglieder:

Projektleiter

  • Dr. Özkan Yildiz

PostDoc

  • Dr. Javier Carrera-Casanova

Ph. D. students

  • Abhishek Acharya
  • Ekaterina Kapotova
  • Katharina van Pee
  • David Wöhlert

Diploma student

  • Katrin Jasmin Jäger

Technical Assistant

  • Heidi Betz
  • Sabine Häder